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A DNA binding loop in the gene 4 helicase‐primase of bacteriophage T7
Author(s) -
Mukherjee Sourav,
Crampton Donald J,
Richardson Charles C
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a910-d
Subject(s) - primase , helicase , biology , replisome , dna , replication protein a , microbiology and biotechnology , dna polymerase , dna replication , dna clamp , ddb1 , hmg box , prokaryotic dna replication , dna polymerase ii , biochemistry , gene , dna binding protein , circular bacterial chromosome , transcription factor , rna , reverse transcriptase
The gene 4 protein encoded by bacteriophage T7 provides both helicase and primase activities at the replication fork. The primase domain is located in the N‐terminal half of the polypeptide and the helicase domain in the C‐terminal half. The gene 4 protein assembles on single‐stranded DNA as a hexamer and then translocates unidirectionally 5′ to 3′ along the strand using the energy of the hydrolysis of deoxythymidine tri‐phosphate (dTTP), thus unwinding double‐stranded DNA (dsDNA) to yield single‐stranded DNA (ssDNA). The crystal structure of the helicase domain of gene 4 protein reveals a flexible segment (residues 464–475), designated as loop II, that lies between conserved motifs H3 and H4. Loop II although not having a defined motif, is distinctly basic with three lysines (K) that protrude into the central DNA‐binding cavity formed by the hexameric protein. It is, therefore, a likely candidate for modulating the binding of gene 4 protein to single‐stranded DNA. Using in vitro mutagenesis we have replaced K467, K471, and K473 with alanines (A). The altered protein lacking basic residues in loop II (gp4‐KloopIIA) does not complement T7Δgp4 infection of E. coli . Although gp4‐KloopIIA formed oligomers equally well as wild‐type protein, it is severely defective in binding to ssDNA. As a consequence, ssDNA‐dependent stimulation of dTTPase activity and DNA unwinding are both abolished. Thus, the basic residues of loop II are involved in the binding of gene 4 protein to single‐stranded DNA. * SM and DJC contributed equally to the work.